Electrophoretic process with continuous scanning



March 11, 1969 R. N. RAND 3,432,414

ELECTRQPHORETIC PROCESS WITH CONTINUOUS SCANNING Filed April 1, 1965 PRESET TEMP. CONTROLLED HEAT RESERVOIR zxcmmeen 2 f |;|]|||I|1., I

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ROYDEN N. RAND INVENTOR AT TORNEYS United States Patent 3,432,414 ELECTROPHORETIC PROCESS WITH CONTINUOUS SCANNING Royden N. Rand, Pittsford, N.Y., assignor to Bausch & Lomb Incorporated, Rochester, N.Y., a corporation of New York Filed Apr. 1, 1965, Ser. No. 444,616 U.S. Cl. 204180 Int. Cl. 301k /00 7 Claims ABSTRACT OF THE DISCLOSURE This invention relates to electrophoresis and more particularly to a method of electrophoresis.

Electrophoresis provides a differential movement of components of a test sample in an electric field. The movement is in proportion to the charge carried, the mobility of the ions, the medium supporting the test sample etc. The process adapts itself well to clinical analysis of enzymes, Serums, proteins, among other mixtures. The process should provide good resolution. Accordingly, this provdies a migration medium which provides a control of the ionic content in the migration medium and causes the various fractions of the test sample to migrate in varying proportions to provide good resolution of the components analyzed in the electrophoresis process.

It is an object of this invention to provide a method of electrophoresis.

It is another object of this invention to provide a method of electrophoresis whereby temperature, humidity, and pH factor of the migration medium are accurately controlled.

It is further object of this invention to provide an electrophoretic process wherein the ionic content of the migration medium is controlled, and the pH factor of the buffer solution is controlled together with temperature and humidity in the migration chamber to rovide improved resolution in the analysis of the sample.

The objects of this invention are accomplished by pro viding a migration tank defining buffer solution chambers and a migration table for supporting a migration medium extending intermediate said chambers. A buffer solution forms a single mass and is confined in the migration tank which is filled to permit an overflow forming a layer extending across the migration table. Although the total mass of the buffer solution contains an agarose of the desired percentage only the portion forming the layer across the migration table provides the migration medium for the sample being analyzed. The portion of the buffer solution contained in electrode chambers is confined to a circuitous path which attenuates the movement of ions from the electrodes to the migration table. This substantially limits the movement of the hydroxyl and hydrogen ions from the electrodes to the migration medium forming the layer on the table intermediate the buffer solutions chambers. In this manner the pH factor of the migration medium supported by the migration table is maintained within fairly close limits.

A cooling fluid is also circulated underneath the migration table to provide a fairly constant temperature of the migration medium. A cover is placed over the migration table during the migration step of the process to control humidity in the enclosed compartment over the migration medium.

Subsequent to the migration of the fractions in the sample a scanning device is used to scan the migrated fractions and a readout system receives an electrical signal which records the results of the analysis responsive absorption or transmission of the migrated sample.

The preferred embodiment of this invention is described in the subsequent paragraphs and illustrated in the attached drawings:

FIG. 1 is a plan view of an electrophoresis apparatus with sections broken away.

FIG. 2 is a cross section view showing a scanning means with the electrophoresis apparatus taken on line 22 of FIG. 1.

FIG. 3 is a similar cross section view to that of FIG. 2 except the apparatus is shown during the migration step of the method.

FIG. 4 is a fragmentary cross section view of FIG. 1 taken on line 44.

Referring to the drawings FIG. 1 illustrates a 'plan view of the electrophoresis equipment with the scanning means removed to more clearly illustrate the relative position of the various parts.

FIG. 3 illustrates the cover on the equipment showing the equipment in the migration step of the process.

The migration tank 1 is constructed with a chamber 2 and a chamber 3 on extreme ends of the tank. The migration table 4 includes the plate 18 which forms the intermediate section of the migration tank. This plate defines a horizontal surface 5 which supports the migration medium 6. The migration medium 6 is an adjoining layer of the electrophoresis medium confined within the chambers 2 and 3 which are filled to a level to overflow the migration table 4. The chambers 2 and 3 receive a plurality of bafiles 7, 8, 9, 10, and 11, 12, 13 and 14 respectively. The bafiles form alternate passages on their upper and lower ends to provide a circuitous path between the electrodes 15 and 16 and the migration layer 6. Effectively these bafiles provide an extension of a thicker layer of the electrophoretic medium than the migration layer 6. By placing the electrodes 15 and 16 at a remote position to the migration layer and causing the hydroxyl ions and hydrogen ions produced by electrolysis to pass a greater distance the pH factor may be controlled in the layer 6. The electrophoretic medium is a solution conitaining a gelling additive which effectively produces a fluid gel on the migration layer which is temperature controlled, but which is primarily in the liquid state in the chambers due to the heating effect of the electric. current passing through the electrophoretic medium.

The table 4 is temperature controlled by a conduit 17 which winds underneath the plate 18 of the migration table 4. The plate 18 is constructed of a light transmitting material such as quartz which will accommodate the passage of light of the bandwidth between the wavelengths of 200 millimicrons and into the visible spectrum. The plate has an upper surface 5 which lies in a horizontal plane and carries the electrophoretic medium 19 which is poured into the chambers 2 and 3 and overflows onto the surface 5 and seeks its own level to form a uniform thickness of the migration layer 6. Intermediate the windings of the conduit 17 but on the upper surface of the plate 18 are optical areas 20 which extend longitudinally across the surface 5 of the plate 18. These areas receive the sample which is migrated in an electric field. Subsequent to the migration these areas can be optically scanned as will be described in a subsequent paragraph.

The temperature of the migration medium 6 is controlled by the fluid passing through the conduit 17 which enters through the tubing 21 from the pump 22. The temperature of the fiuid is controlled by the heat exchanger 23 as it is received from the reservoir 24 which is connected through the tube 25 to the outlet of the conduit 17. The heat exchanger is provided with means for presetting the temperature of the fluid to the desired temperature.

The applicator 26 includes a bar 27 which is positioned on the edges 28 and 29 of the migration tank and carries a string 49 which is stretched across the lower ends of the slotted blocks 50 extending into the tank 1. Subsequent to applying the sample to the surface of the migration medium the cover 30 is positioned over the migration table. The cover eifectively provides an enclosed compartment in the area of migration when the sample is migrated into its various fractions due to the electric field.

A source of direct current voltage 31 is connected through the switch 32 to the terminals 15 and 16. The voltage may be set at any voltage between 0 and 500 DC. The electric field is created over the migration surface through the electrophoretic medium when the switch 32 is closed. The field is maintained across the migration layer for a predetermined time sufficient to migrate the various fractions of the sample.

Subsequent to the migration of the sample the scanning device is used to provide a readout of the presence and the magnitude of the various fractions migrated. The inventor does not intend to limit his invention to the scanning device illustrated. The scanning device shown includes a motor 33 with a driving means 34 connected to the threaded shaft 35 supported in the bearings 36 and 37. The carriage 38 is threaded internally and moves longitudinally on the shaft 35 as the shaft is rotated. The carriage 38 supports a source of radiation in the monochromator 39 directing radiation of the desired wavelength through the slit and the optical system toward the photosensor 40. A suitable electrical means of energization is connected to the monochromator 39 for energizing the source of radiation to produce exit radiation of the desired wavelength. The optical system images the slit on the migration medium supported by the plate 18. The photosensor 40 receives a radiation signal and generates an electrical signal which is conducted through the conductors YY to the readout recorder 41. The readout recorder may be any suitable means such as a meter, a recording instrument etc. which gives a visible indication or a record or any readout desired.

The process will be described in the following v paragraphs.

The buffer solution for use in electrophoresis processes may be sodium phosphate or potassium phosphate or any other buffer solution which has a suitable pH factor. Phosphates are useful in this process because they transmit light in the ultraviolet range. The pH factor of approximately 8.8 operates satisfactorily for serum proteins. Butter solutions have been used in this process which have pH factors which range from 1.7 to .9 for other separation processes. The bulfer solution contains a percentage of sodium or potassium phosphate in water. A satisfactory range of the sodium hydroxide would be .6 to .7 gram per 100 milliliters and for the potassium phosphate approximately .5 to .6 gram to 100 milliliters.

The agarose percentage by volume may be .1 to .3 gram per 100 milliliters. The inventor does not Wish to limit the butter solution to any specific range nor to the specific compounds mentioned but is merely setting forth an illustration which will satisfactorily operate in this electrophoretic process.

The electrophoretic medium containing water, phosphate, and agarose is poured into the chambers 2 and 3 until the medium overflows the surface 5 of the migration table to a thickness of approximately 1.7 mm. The horizontal surface 5 of the table 4 supports a migration layer. The upper surface of this layer is controlled by the level of the electrophoretic medium due to the force of gravity.

A sample is then positioned on the string 49 which is stretched across the applicator 26 and then positioned on the surface or in the migration layer. A plurality of samples may be deposited on the surface of the migration layer in accordance with the number of optical areas 20 formed on the migration table 4. Subsequently to depositing the samples on the migration layer the switch 32 is closed which applies a voltage across the terminals 15 and 16. This in turn generates an electric field across the layer 6. The various fractions in the sample will then migrate in accordance with their mobilities, charges, and physical characteristics etc. The electric field is generated across the migration layer for the predetermined time to provide the desired migration.

Subsequent to migration of the various fractions the cover 30 is then removed and the scanning means is positioned to scan each of the optical areas 20 sequentially. The radiation from the source of monochromator 39 is directed through the sample on the migration layer supported by the plate 18 and a signal responsive to light transmission passing through various fractions carried on or in the medium 6 supported by the plate 18 is generated by the photosensor 40. A suitable readout recorder then records the evaluation determined by the scanning means. The readout may have an indication of absorption or transmission.

The preferred embodiment of this invention has been illustrated and described and it is understood that other embodiments of this invention may be devised which would fall within the scope of the invention which is defined by the attached claims.

I claim:

1. A method of making electrophoresis separations and measurements thereof comprising the following steps, confining a mass of an electrophoretic medium into two spaced bodies with a'thin horizontal layer of said medium defining a migration layer interconnecting said bodies to exhibit a common surface level, depositing a sample on the upper surface of the migration layer, applying a direct current voltage to the two spaced bodies remote from the migration layer to generate an electric field across the migration layer for a predetermined time to migrate the various fractions of the sample while maintaining the pH factor constant, scanning the migrated fractions with radiation directed through said migration layer, sensing the absorption of radiation by the various fractions, and generating an electrical output signal responsive to the magnitudes of absorption of the migrated fractions thereby electrophoretically analyzing a sample deposited on the migration layer.

2. A method of making electrophoresis separations and measurements thereof comprising, confining two bodies of a buffer solution including agarose in spaced relation to each other with a thin horizontal layer of said buffer solution interconnecting said two bodies to exhibit a common surface layer, depositing a test sample on the surface of the migration medium, maintaining a constant humidity and temperature of the migration medium, applying a DC voltage at points on the two bodies remote from the horizontal layer to generate an electric field across said migration layer to migrate the various fractions of the sample, directing a radiation through the migrated fractions and the migration medium and scanning the length of migration of said fractions, sensing the absorption of radiation in response to the presence and amount of the various fractions, and generating an electrical readout signal to record the analysis in said electrophoresis process.

3. A method of making electrophoresis separations and measurements thereof comprising the following steps, adding agarose in a proportion by volume of .1 to .3 gram per milliliter to a buffer solution having an approximate pH factor of 8.8 to form an electrophoretic medium, confining said electrophoretic medium in two bodies spaced relative to each other with a thin horizontal layer of said medium defining a migration surface interconnecting said spaced bodies to exhibit a common surface level, depositing a test sample on the migration surface, maintaining a constant humidity and temperature of the migration medium while applying a voltage across the two bodies to migrate the fractions of the test sample, subsequently directing a radiation through the migrated sample and medium and scanning the length of the migrated fractions, simultaneously sensing the absorption of radiation by the fractions of the sample, and generating an electrical signal responsive thereto to provide an electrophoresis analysis. I

4. A method of making electrophoresis separations and measurements thereof comprising the following steps, adding agarose in the amounts of .1 to .3 gram per milliliter of a buffer solution to form the electrophoresis medium, confining the electrophoresis medium in two bodies in spaced relation to each other with a thin horizontal layer of said medium defining a migration layer interconnecting said bodies to exhibit a common surface level, depositing a test sample on the surface of said layer, maintaining a constant humidity and temperature of the migration medium while simultaneously applying a direct current voltage to the bodies of the electrophoresis medium to migrate the various fractions in the sample, subsequently directing monochromatic radiation through the portion of the migration medium carrying the migrated sample and scanning the length of the migrated sample, sensing the presence and the amount of the migrated fractions in the sample and generating an electrical signal for re cording the analysis of the electrophoresis process.

5. A method of making electrophoresis separations and measurements thereof comprising the following steps, adding agarose in the amounts within the range of .1 to .3 gram per milliliter to a phosphate bulfer solution to form an electrophoresis medium, confining the electrophoresis medium into a thin horizontal layer of migration medium interconnecting two relatively thicker layers to exhibit a common surface level, depositing a test sample on the surface of thin horizontal layer, maintaining a constant humidity and temperature of the migration medium while simultaneously applying a voltage on the extreme ends of the relatively thicker layers of said electrophoresis medium for a time suflicient to migrate the various fractions in the test sample to provide good resolution, directing a monochromatic radiation of approximately 200 millimicrons wavelength through the length of the migrated sample on said migration layer, sensing the presence and the amount of the various fractions in said migrated sample, and generating an electrical signal and a readout responsive there to and providing an analysis of said sample.

6. A method of making electrophoresis separations and measurements thereof comprising the following steps, add

ing an agarose in the amounts of .1 to .3 gram per milliliter to a phosphate bulfer solution to form an electrophoresis medium, confining the electrophoresis medium to a thin horizontal migration layer interconnecting two relatively thicker layers to exhibit a common surface level, depositing a sample on the surface of said migration layer, applying a direct current voltage to the extreme ends of said thicker layers for migrating the various fractions on the surface of said migration medium and controlling the pH factor on the migration layer by the remote connection of said electrodes, maintaining a constant temperature and humidity on the migration medium, subsequently directing monochromatic radiation through the migration medium and the migrated sample and scanning a length of the medium for a distance at least equal to the length of migration of the fractions of said sample, sensing the radiation absorption by the fractions in said migrated sample and generating an electrical signal responsive thereto for providing a readout of the analysis provided in said electrophoresis process.

7. A method of making electrophoresis separations and measurements thereof comprising the following steps, add ing agarose in a proportion by volume of .1 to .3 gram per milliliter of a butter solution having a pH factor within the range of 1.7 to 9 for forming an electrophoretic medium, confining said electrophoretic medium in two bodies spaced relative to each other with a thin horizontal layer of said medium defining a migration surface interconnecting the two bodies to exhibit a common surface level, depositing a test sample on the migration surface, maintaining a constant humidity and temperature of the migration medium while applying a voltage across the two bodies to migrate the fractions of the test sample, subsequently directing a radiation through the migrated sample and medium and scanning the length of the migrated fractions, simultaneously sensing the absorption of radiation by the fractions of the sample, and simultaneously generating an electrical signal responsive thereto to provide a readout of the electrophoresis separation.

References Cited UNITED STATES PATENTS 2,843,540 7/1958 Ressler 204-480 3,047,489 7/1962 Raymond 204-299 3,129,158 4/1964 Raymond et al 204- OTHER REFERENCES Hjertn: Zone Electrophoresis in Columns of Agarose Suspension, Journal of Chromatography, p. 510, vol. 12, No. 4, December 1963.

JOHN H. MACK, Primary Examiner.

A. C. PRESCOTT, Assistant Examiner.

U.S. Cl. X.R. 204'299 

